Biochemical and Physicochemical Properties of Actomyosin from Pre- and Post-spawned Hake (Merluccius hubbsi) Stored on Ice

Biochemical and physicochemical properties of natural actomyosin (NAM) from pre- and post-spawned hake stored on ice were studied. At the beginning of storage both reduced viscosity and ATPases activities of NAM from post-spawned hake were about 3 times higher than those of the pre-spawned ones, except for the Mg²⁺-(Ca²⁺)-ATPase. Higher loss of functional components during pre-spawned hake NAM purification was found compared to NAM from post-spawned fish. Reduced viscosity and Mg²⁺-(EGTA)-ATPase activity of NAM from both pre- and post-spawned hake decreased following ice storage. SDS-PAGE patterns of NAM from pre-spawned hake showed an absence of the myosin heavy chain and the presence of a 160 kDa component at zero time of storage. No proteolysis occurred in ice-stored fish caught in both gonadal stages.     

Influence of Gonadal Stage of Hake (Merluccius Hubbsi Marini) on Biochemical Properties of Myofibrils Stored at 2 to 4 °C

ABSTRACT: The influence of the gonadal stage of hake on the biochemical properties of myofibrils stored at 2 to 4 °C was studied. At 0 time and during storage, both Mg²⁺-Ca²⁺-ATPase activity and Ca²⁺ sensitivity of myofibrils from post-spawned hake were significantly higher (p < 0.01) than those of pre-spawned fish. The profiles of SDS-PAGE gels of unstored and stored myofibrils from pre-spawned hake showed a partially denatured myosin heavy chain. The actin-myosin ratio in myofibrils from pre-spawned hake was significantly lower (p < 0.01) than the ratio in post-spawned hake. Irrespective of the gonadal condition of fish, no changes in the myosin-actin ratio of stored myofibrils were observed.     

Biochemical and Physicochemical Properties of Actomyosin from Frozen Pre- and Post-spawned Hake

Protein solubility, reduced and intrinsic viscosity, and enzymatic activities of actomyosin showed that frozen fillets from pre-spawned hake, stored at — 20°C, deteriorated faster than those from post-spawned hake. The post-spawned actomyosin showed an 18% increase in reduced viscosity, a larger hydrophilic surface, and an increase (P<0.001) in Mg²⁺-, Mg²⁺-EGTA- and Ca²⁺-ATPase activities. Protein solubilities of 85.5 52.5 ± and 33 ± 2.0% were obtained at the end of storage for post and pre-spawned fillets, respectively. Enzymatic activities of post-spawned fillets persisted up to 60 days. No changes were detected in characteristic polypeptide bands of actomyosin or in the myosin/actin ratio related to either gonadal condition during storage.     

Proteolytic Activity of Muscle in Pre- and Post-Spawning Hake (Merluccius hubbsi Marini) After Frozen Storage

The proteolytic activity of fillets from fresh fish and after frozen storage of hake muscle from pre- and post-spawned fish was investigated. The autolytic activity of fresh muscle extracts from pre-spawned muscle extracts was significantly higher than that of post-spawned, except for pH 3.4 and incubation temperature of 60 °C where no significant difference was found. In both gonadal conditions, the freezing of hake fillets affected the proteolytic activity of crude muscle extracts. After 50 d of frozen storage, the proteolytic activity values of crude muscle extracts remained higher than the fresh values, except for pre-spawned extracts incubated at 37 °C. At longer storage period (110 d), the proteolytic activity continued with similar or higher values compared with those observed for fresh muscle extracts. Densitometric analysis of crude muscle extracts indicated degradation of MHC in fresh and frozen storage at both gonadal conditions under incubation conditions of pH 6.2 and temperatures 37 and 60 °C, pH 8.5 and temperatures 37 and 60 °C, pH 3.4 and temperature 4 °C.     

Comparison of gilthead sea bream (Sparus aurata) and hake (Merluccius merluccius) muscle proteins during iced and frozen storage

Gilthead sea bream (Sparus aurata) and hake (Merluccius merluccius) muscle behave differently during storage, whether in ice or deep frozen. Rapid changes have been observed in the texture of hake muscle during frozen storage, while gilthead sea bream has proved to be more stable. In order to ascertain the role of muscle proteins in the changes observed during storage, parameters related to protein functionality and the properties of extracted natural actomyosin (NAM) were studied initially and during storage in ice or at ?20 °C. Initially, the parameters related to functionality had higher values in hake muscle and extracted NAM than in gilthead sea bream. At the end of iced storage (22 days), less myosin heavy chain (MHC) and actin were extracted from hake, but there was practically no change in gilthead sea bream. This decrease was not accompanied by lower Ca²⁺‐ATPase activity. Freezing produced no drastic changes, with lower values for gilthead sea bream. However, this species was more stable after 1 year, except for the Ca²⁺‐ATPase activity of NAM. This suggests that the changes that hake proteins underwent during storage particularly affected properties related to aggregation, whereas in gilthead sea bream the changes hardly affected the formation of soluble or insoluble aggregates but did affect the active sites of myosin. © 2002 Society of Chemical Industry     

Cross-Linking of Myosin Heavy Chains from Cod, Herring and Silver Hake During Thermal Setting

Cross-linking of myofibrillar proteins extracted from cod (Gadus morhua), herring (Clupea harengus) and silver hake (Merluccius bilinearis) was studied in 0.6M NaCl, pH 6.5 at 40°C and evaluated turbidimetrically and by SDS polyacrylamide gel electrophoresis coupled with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a zero-length crosslinker. Turbidities of heat-treated cod and silver hake myofibril/myosin solutions were significantly higher than those of herring. Electrophoretic results showed that the myosin heavy chain (MHC) was the principal myofibrillar protein cross-linked to form a polymerized complex during the heat treatment. Cross-linking ability of MHC from the three fish species was different; herring MHC formed only small polymers (n≦3) but cod and silver hake MHC formed both small and large polymers (n≦6).     

Biochemical and physicochemical properties of actomyosin from pre- and post-spawned flounder (Paralichtys patagonicus) stored on ice

The biochemical and physicochemical properties of actomyosin from pre- and post-spawned flounder stored on ice were studied. Irrespective of the gonadal condition of the fish, a gradual decrease in reduced viscosity, Mg²⁺ATPase and Mg²⁺Ca²⁺ATPase activities of actomyosin was observed. A similar decrease in the Mg²⁺ EGTA ATPase activity of actomyosin from post-spawned flounder was also observed. The decrease in enzymatic activities was accompanied by an increase in surface hydrophobicity of the protein. No signs of proteolysis of the major components of the actomyosin complex were detected. The relative percentage of myosin decreased and that of actin increased in actomyosin from pre- and post-spawned flounder stored on ice. These changes occur earlier in actomyosin from prespawned flounder. The results of this paper indicate that actomyosin from flounder denatured during fish storage on ice and suggest that this denaturation is due to structural and conformational changes in myosin which led to aggregation of this protein.     

Thermal Denaturation in Fish Muscle Proteins During Gelling: Effect of Spawning Condition

We studied thermal denaturation of myofibrillar proteins from pre-and post-spawning hake by differential scanning calorimetry (DSC), and evaluated denaturation kinetics under both conditions. The denaturation enthalpies of all pre-spawning fish muscle extracts were less than those from post-spawning. The area under the DSC thermogram corresponding to myosin denaturation was smaller in myofibrillar extracts from pre-spawning than from post-spawning hake, while the areas corresponding to denaturation of actin were similar. Between 40 and 55°C the myosin denaturation rates were greater for post-spawning than for pre-spawning hake. Both entalphies and kinetic data indicated proteins of fish in a better biological condition (post-spawned) denature more rapidly and completely.     

Chemical and Physical Changes in Red Hake Fillets During Frozen Storage

Changes in chemical and physical properties of frozen red hake fillets were determined as a function of time. The decrease in protein extractability could be partly reversed by mercaptoethanol. Sodium dodecylsulfate greatly reduced the loss in extractability. Polypeptides that would not enter 10% polyacrylamide gels after treatment with SDS and mercaptoethanol were formed during frozen storage. The rate of decrease of the myosin heavy chain followed first order kinetics with a rate constant of –0.054 per week at –7°C and closely paralleled the appearance of the “cross-linked” peptides. Uniaxial compression moduli correlated well with the dimethylamine produced and the loss of the myosin heavy chain after SDS-PAGE.     

Conformational Change in Actomyosin from Post-spawned Hake Stored on Ice

The changes that lead to a decrease in the viscosity of actomyosin of post-spawned hake stored on ice were investigated. Surface hydro-phobicity of actomyosin from hake increased significantly in the first 3 days of storage. Compared with that stored 3 days, the viscosity of actomyosin from day 0 was greater and its changes suggested greater hydrophylic surface area. Electron microscopy showed changes in the filamentous structure of actomyosin during ice storage. Actomyosin from hake stored in ice is hypothesized to undergo conformational changes including exposure of hydrophobic groups resulting in drastic changes in hydrodynamic properties and a loss of the characteristic arrowhead structure of fresh acotmysin.     

Effect of ageing on the properties of myofibrils of rabbit muscle

The effect of ageing of rabbit muscle at 4° and 15–18° on the extractability and adenosine triphosphatase activity of the myofibrils has been examined. The amount of protein extracted by M-KCL-4 mM sodium glycerophosphate (pH 6.2) and by 0.1 M sodium tetrapyrophosphate-4 mM MgCl₂-10 mM-KH₂PO₄ (pH 7.2) increased as the muscle aged. By using the amount of Ca²⁺ adenosine triphosphatase extracted, i.e. the enzyme associated with myosin, as a measure of the amount of myosin in the actomyosin extracted, it was possible to show that the buffered potassium chloride did not extract all the actomyosin from the myofibrils of pre-rigor muscle. As the muscle aged, more actomyosin was extracted, together with some tropomyosin. Pyrophosphate, however, extracted all the myosin from the pre-rigor muscle, and the increase in the protein extracted from aged muscle was due to actin and tropomyosin in addition to myosin. It is suggested that these changes are caused by a disruption of the Z-band structure during ageing, perhaps due to the hydrolysis of tropomyosin by proteolytic enzymes. The specific Ca²⁺ adenosine triphosphatase activity of the myofibrils was unaltered by ageing but the specific Mg²⁺-activated adenosine triphosphatase, i.e. the enzyme associated with actomyosin, was reduced by about one-third. This latter result may be caused by a change in the mode of linkage of actin to myosin.     

Extractability and thermal stability of frozen hake (Merluccius merluccius) fillets stored at −10 and −30 °C

The relationship between thermal stability changes and functionality loss was monitored in hake muscle fillets stored for 40 weeks at ?10 and ?30 °C. The evolution of changes in apparent viscosity, dimethylamine formation and extractability of muscle proteins in NaCl, sodium dodecyl sulphate (SDS) or SDS plus mercaptoethanol showed drastic differences as a function of temperature. At the higher storage temperature, both myosin heavy chain and collagen were the most severely unextracted in salt and SDS solutions, with actin becoming unextractable at the end of storage. Differential scanning calorimetry showed differences with storage time and temperature in both onset temperature and thermal denaturation enthalpy, mostly affecting the myosin transitions. Some protein denaturation occurred with little or no functionality loss. A considerably high fraction of hake muscle proteins remained in the native‐like condition even at the higher frozen storage temperature. In these conditions both apparent viscosity and myosin and actin extractability in NaCl were very low. © 2002 Society of Chemical Industry     

Post-mortem changes of muscle from fresh water prawn (Macrobrachium rosenbergii) as influenced by spawning stages

Post-mortem changes of the muscle from pre- and post-spawned fresh water prawn (Macrobrachium rosenbergii) were comparatively monitored during 7 days of iced storage. During the storage, the muscle of pre-spawned prawn had a greater value of trichloroacetic acid (TCA) soluble peptide, heat soluble collagen and pepsin-soluble collagen (PSC) contents than did post-spawned counterpart. Those components in the muscle of both prawns increased markedly after 3 days of storage (p < 0.05). Conversely, insoluble collagen (ISC) content, shear force value and texture liking of both prawns decreased (p < 0.05), indicating the softening of muscle. No changes in protein patterns were observed, except the decreased band intensity of 66 kDa protein in water soluble fraction of both prawns was found after 3 days of storage. T_max and enthalpy of PSC from both prawns decreased during the first 4 days of storage (p < 0.05), suggesting the degradation or denaturation of collagen in the muscle. Light microscopic study showed the lowering of intercellular connection of raw meat and higher gaping in cooked meat when the samples were stored for a longer time. Therefore, post-mortem characteristics of prawn muscle was affected by storage time and spawning stages.     

Effects of previous frozen storage on chemical, microbiological and sensory changes during chilled storage of Mediterranean hake (Merluccius merluccius) after thawing

The present work aimed to investigate the influence of freezing (−20 °C for 6 months) on the susceptibility of hake to chemical, microbiological and sensory spoilage once thawed and stored in ice. Thus, volatile and biogenic amines, counts of psychrotrophic and mesophilic bacteria, enterobacteria, pseudomonads and Shewanella together with sensory evaluation (of raw and cooked samples) were monitored during 14 days of iced storage in both fresh and frozen-thawed hake (Merluccius merluccius). At the beginning of ice storage, freshness parameters were equivalent in both batches (fresh and frozen-thawed hake), but different behaviour was observed during the iced storage. Overall, levels of volatile and biogenic amines increased much later and then decreased to lower values in frozen-thawed hake in accordance with the delay of microbial development in comparison with fresh hake. On the contrary, sensory spoilage occurred earlier in frozen-thawed hake. Therefore, the usual accepted or regulated limits of acceptability of chemical and microbiological parameters would not be suitable for freshness assessment of frozen-thawed hake.     

Myosin heavy chain degradation during post mortem storage of Atlantic cod (Gadus morhua L.)

Post mortem proteolytic degradation of fish fillets leads to textural changes like muscle softening and gaping. In this study proteolytic degradation of myosin heavy chain (MHC) was monitored during storage of muscle and of isolated myofibrils at different temperatures and pH-values by the use of MHC-specific antibodies. The ability of cathepsin D to associate to myofibrillar proteins was also studied. Muscle stored at 6 °C and isolated myofibrils stored at 0 °C, 6 °C and 20 °C were degraded at pH 6.3 or lower. Cathepsin D could be found associated with extensively washed myofibrils. Inhibition of cathepsin D during storage affected the observed MHC-degradation at pH 5.5, but not at pH 6.3. This indicates that cathepsin D to a less extend than formerly believed, is responsible post mortem degradation of MHC.     

Cathepsin Degradation of Pacific Whiting Surimi Proteins

Cathepsin B was the most active cysteine protease in Pacific whiting fish fillets; cathepsin L was predominant in surimi. Cathepsin L showed highest activity at 55°C in both fish fillets and surimi, indicating its function in myosin degradation during conventional heating of fillets and surimi, gels. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Myosin heavy chain was the primary substrate during autolysis of surimi paste and actin and myosin light chain showed limited hydrolysis during 2 hr incubation. Purified Pacific whiting cathepsin L hydrolyzed myofibrils, myosin and native and heat-denatured collagen. The degradation pattern of myofibrils by the protease was the same as the autolytic pattern of surimi.     

Effect of frozen storage of hake,sardine and mixed minces on natural actomyosin extracted in salt solutions

Natural actomyosin extracted in salt solutions from mixtures of hake and sardine minces (3:1; 1:1 and 1:3 w/w) stored frozen for up to 1 year differed in the amount extracted and in the characteristics of the extracts. In the mixed minces the amount of natural actomyosin extracted decreased during frozen storage at a higher rate than that theoretically corresponding to the amount of hake in the mixes. With increasing storage time and proportion of sardine a lower percentage of myosin heavy chain and actin was observed by electrophoresis. An increased size of aggregates was also observed by electrophoresis and transmission electron microscopy. The stability of emulsions was enhanced when aggregates appeared in the extracts. The decrease in the amount of natural actomyosin extracted does not explain the changes observed in the texture of the minces during frozen storage. This may indicate that the size of the aggregates unextractable in salt solutions, independently of the type of bonds that bind the proteins in the aggregates, plays an important role in the textural changes observed. Copyright © 2003 Society of Chemical Industry     

ABSENCE OF PROTEOLYSIS IN PURIFIED MYOFIBRILS OF POSTSPAWNED HAKE (MERLUCCIUS HUBBSI MARINI) DURING IN VITRO STORAGE AT 37C

The biochemical behavior of myofibrils from postspawned hake during in vitro storage at 37C was investigated. SDS‐PAGE, densitometric analysis of the band areas corresponding to the major myofibrillar proteins, and TCA soluble peptides determination showed no evidence of proteolysis in myofibrils after 44 h of incubation either in presence or in absence of a cocktail of protease inhibitors (1 mMPMSF+1mMiodoacetic acid + 1mM of EDTA). The absence of proteolysis in stored postspawned hake myofibrils contrasts with the autolysis in those from prespawned fish previously reported, indicating an influence of the reproductive cycle on the proteolytic activity closely associated to myofibrils.     

Effect of Cathepsin D on Bovine Myofibrils under Different Conditions of pH and Temperature

Purified cathepsin D was incubated with bovine skeletal muscle myofibrils under in virro conditions resembling those found in postmortem muscle. SDS-PAGE analysis of myofibrils treated at pH 5.5 and 37°C and the sedimented, showed degradation of myosin heavy chains and titin. A small amount of actin, tropomyosin, troponins T and I, and myosin light chains also were degraded. The cathepsin D treated myofibrils were not fragmented to any greater extend than untreated myofibrils. Raising the pH and/or lowering the temperature greatly reduced the effectiveness of cathepsin D suggesting that the enzyme does not play a principal role in the tenderization process occurring in muscle postmortem.     

The Pepsin Digestibility of Thermal Gel Products Made from White Croaker (Pennahia argentata) Muscle in Associating with Myosin Polymerization Levels

Thermal gels were made from white croaker (Pennahia argentata) surimi at various polymerization levels of myosin heavy chains induced by suwari treatment at 38 °C for various time periods and subsequently heated at 85 °C for 20 min. Myosin heavy chain polymerization levels were also achieved in the presence of microbial transglutaminase (MTG) added at various concentrations in the surimi. The breaking strength and breaking strain rate were markedly increased during suwari treatment up to 60 min in accordance with the increased levels of myosin heavy chain polymerization. MTG enhanced myosin heavy chain polymerization during suwari treatment for 15 and 30 min, resulting in the increase of breaking strength. The solubilization in 8 M urea and pepsin digestibility of these gels as well as angiotensin I‐converting enzyme (ACE) inhibitory activity of their pepsin digests were decreased with the increased levels of myosin heavy chain polymerization. These results suggest that myosin heavy chain polymerization affects not only rheological properties of thermal gels but also their functional properties for human health.